1. Field of the Invention
This invention relates to murine hybridoma cell lines and monoclonal antibodies produced therefrom which may be used to detect severe forms of the citrus tristeza virus in citrus plant tissue by immunological assay.
2. Description of the Prior Art
A foreign entity such as a virus can be used to provoke an immune response in a mouse. Antibody-producing white blood cells of the mouse react to many specific sites on the virus, called antigenic determinants. By selecting a white blood cell that produces antibody to an antigenic determinant present only on one type or class of virus, a researcher is able to develop an immunological probe which can identify that type of virus without reacting to all other viruses. The antibody-producing cell is fused with a cancer cell (myeloma), resulting in a new cell that retains the properties of both parent cells. The new cell (hybridoma) still produces the desired antibody, but now is immortal like the cancer cell. In practical terms, this means that the hybridoma is a constant and unlimited source of a single type of antibody that never varies qualitatively. Hybridomas that have been selectively cloned several times are called monoclonal antibodies [Goding, "Monoclonal Antibodies: Principles and Practice," pp. 5-40, Academic Press Inc., London (1983)].
Citrus tristeza virus (CTV) is the cause of severe stem pitting and decline diseases of citrus and is one of the most economically important citrus pathogens worldwide [Bar-Joseph et al., Proc. Intern. Soc. Citriculture 1: 419-422 (1981); Whiteside et al. (eds.), "Compendium of Citrus Diseases," pp. 48-50, APS Press, St. Paul, Minn. (1988)]. There is great diversity of symptoms induced among different isolates of CTV, and symptom severity is often host specific [Garnsey et al., Phytophylactica 19: 151-157 (1987)]. Currently, severity of a given isolate can only be determined by inoculating differential indicator plants or the commercial host [Garnsey et al., supra (1987)]. This is time consuming and not satisfactory for many applications.
Serological tests for CTV have been developed [Brlansky et al., Proc. 9th Conf. Intern. Organ. Citrus Virol., Garnsey et al. (eds.), pp. 337-342, IOCV, Gainesville, Fla. (1984); Garnsey et al., Phytopathology 68: 88-95 (1979)], and enzyme-linked immunosorbent assay (ELISA) tests have been widely used for survey, certification, and eradication work as well as for research [Cambra et al., Proc. Intern. Soc. Citriculture 1: 444-448 (1982); Garnsey et al., Proc. Intern. Soc. Citriculture 1: 448-452 (1981); Ke et al., Proc. 9th Conf. Intern. Organ. Citrus Virol., Garnsey et al. (eds.), pp. 70-75, IOCV, Riverside, Calif. (1984)]. Polyclonal antisera have been made to different isolates in several animal species [Garnsey et al., supra (1981)], and a monoclonal antibody also has been produced and made available commercially [Vela et al., J. Gen. Virol. 67: 91-96 (1986)]. The polyclonal and monoclonal antibodies reported are all reactive to a wide range of CTV isolates of differing severity [Garnsey et al., Phytopathology 75: 1311 Abstract (1985); Vela et al., supra (1986)]. These immunological probes are useful for general detection of CTV infection, but do not provide information about biological severity.
The lack of evidence for serological diversity among CTV isolates is consistent with analysis of peptide digests of different isolates which also indicated only small differences in coat protein chemistry among the isolates evaluated [Lee and Calvert, Phytophylactica 19: 205-210 (1987)]. Other approaches, such as cDNA probes [Rosner et al., Phytopathology 76: 820-824 (1986)] and dsRNA analysis [Dodds et al., Phytophylactica 19: 131-137 (1987)], so far lack the desired specificity or reliability, or are not adaptable for rapid, large-scale assays. There is a need for a rapid diagnostic procedure to identify specific severe isolates of CTV. Brlansky et al. [supra] and Vela et al. [Proc. 10th Conf. Intern. Organ. Citrus Virol., Timer et al. (eds.), pp. 55-61, IOCV, Riverside, Calif. (1988)] have suggested that multiple epitopes exist in the CTV coat protein based, respectively, on results obtained with different polyclonal antisera and with different monoclonal antibodies. Vela et al. [supra (1988)] also suggested these epitopes were common to all CTV isolates tested. However, evidence that some epitopes may be strain specific was obtained with polyclonal antiserum to T-36 that showed greater avidity to homologous antigens than to heterologous antigens, whereas antiserum to CTV isolate T-4 reacted equally with both antigens.